CN113122531A - Efficient microbial agent for solving continuous cropping obstacles - Google Patents
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Abstract
The invention discloses a microbial agent, and particularly relates to a high-efficiency microbial agent for solving continuous cropping obstacles. The efficient microbial agent for solving the continuous cropping obstacle consists of a zymocyte liquid and a carrier, wherein the carrier is a functionalized calcium carbonate microsphere prepared by a specific method. The efficient microbial agent for solving the continuous cropping obstacles disclosed by the invention is simple and feasible in preparation process, low in price of raw materials, green, nontoxic and degradable, and can be used for not only solving the continuous cropping obstacles, but also promoting the growth and development of crops, increasing the content of beneficial substances in soil and improving the soil.
Description
Technical Field
The invention relates to the technical field of microbial agents, in particular to a high-efficiency microbial agent for solving continuous cropping obstacles.
Background
Continuous cropping obstacles, also known as continuous cropping diseases, replanting diseases and soil contraindications, refer to the phenomena of poor crop growth, reduced product quality, deteriorated growth status and the like in subsequent planting after the same crop or related crops are planted in the same land even if normal management measures are taken. In crop cultivation, continuous cropping obstacles of crops are very common, and serious continuous cropping obstacles exist in horticultural plants, economic plants, medicinal plants, melons and fruits, vegetables, flowers and the like.
The formation and aggravation of the continuous cropping obstacle of the crops are the external manifestations of the interaction results of the crops and various factors such as soil, microorganisms and the like related to the crops, and are a complicated overall problem. The causes of continuous cropping obstacles of different crops are very different, but the main causes include factors such as deterioration of soil physicochemical properties (nutrient loss, soil acidification, enzyme activity reduction, soil salinization and the like), change of microbial community structure, allelopathy and self-toxicity. At present, the main mode for overcoming the continuous cropping obstacles is chemical control, and although the mode has a certain effect, the mode can cause pollution to the soil environment, and in the long run, adverse effects are generated on the safe and stable production of crops.
The microbial agent is a live bacterial preparation which is prepared by processing a porous substance as an adsorbent after adsorbing a thallus fermentation liquid, can provide nutrients required for growth of crops when being applied to soil, and can also convert or degrade the toxicity and concentration of toxic and harmful substances contained in the soil for planting the crops, thereby playing a role in bioremediation of the soil for planting and solving the problem of continuous cropping obstacles generated by planting the crops.
The invention patent with application number 201610631659.6 discloses a composite microbial inoculum composition capable of reducing crop continuous cropping obstacles, which comprises a solid fertilizer prepared by directly spraying a microbial inoculum and synergistic substances on earthworm dung, wherein the microbial inoculum can be adsorbed on the earthworm dung by spraying, but the adsorption amount is extremely low, and the microbial inoculum and the synergistic substances are easy to fall off in the subsequent drying, transportation and other processes, so that a series of problems of low utilization rate, unobvious use effect and the like of the composite microbial inoculum composition are caused.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a high-efficiency microbial agent for solving continuous cropping obstacles.
In order to solve the technical problems, the invention adopts the technical scheme that:
an efficient microbial agent for solving continuous cropping obstacles comprises a zymocyte liquid and a carrier.
An efficient microbial agent for solving continuous cropping obstacles, which consists of a zymocyte liquid and a carrier according to the mass ratio of 1 (1-3).
The preparation method of the efficient microbial agent for solving the continuous cropping obstacle comprises the following steps: mixing the zymocyte liquid and the carrier according to the mass ratio of 1 (1-3), stirring at the speed of 600-1000rpm for 20-40min, and drying at the temperature of 40-45 ℃ for 4-6h to obtain the high-efficiency microbial agent for solving the continuous cropping obstacle.
The preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain picked by the inoculating loop into a liquid seed culture medium, and culturing at constant temperature of 26 ℃ and 150-180rpm until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 20-26 parts of glucose, 15-20 parts of ammonium sulfate, 10-14 parts of beef extract powder, 6-9 parts of yeast powder, 2-4 parts of potassium chloride, 18-25 parts of ferrous sulfate and 80-90 parts of water, wherein the pH value is 3-3.5, and the beef extract is sterilized at the temperature of 120-;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 8-12% (V/V), and culturing at constant temperature of 26 ℃ and under the condition of 150-180rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 45-55 parts of molasses, 14-18 parts of bran, 30-35 parts of ammonium chloride, 10-14 parts of corn steep liquor, 21-24 parts of beef extract powder, 15-20 parts of yeast powder, 5-10 parts of sodium chloride, 7-10 parts of magnesium sulfate, 10-15 parts of dipotassium hydrogen phosphate, 48-57 parts of ferrous sulfate and 260 parts of water, wherein the pH value is 3-3.5, and the materials are sterilized at 125 ℃ for 15-25 min.
The strain is thiobacillus thiooxidans.
Thiobacillus thiooxidans, gram-negative bacteria, can release organic acids (such as tartaric acid, acetic acid and oxalic acid) to the outside of cells by secreting phytase, nuclease, dehydrogenase, phosphatase and the like so as to acidify insoluble phosphorus and generate available phosphorus which is beneficial to plant absorption.
The carrier is one of diatomite, zeolite, medical stone, calcium carbonate microspheres and functionalized calcium carbonate microspheres.
Preferably, the carrier is calcium carbonate microspheres or functionalized calcium carbonate microspheres.
The preparation method of the calcium carbonate microspheres comprises the following steps: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain the calcium carbonate microspheres.
Most preferably, the carrier is functionalized calcium carbonate microspheres.
Calcium carbonate is an inorganic functional synergistic substance, has the characteristics of low price, environmental friendliness, high stability and the like, and is favorable for improving the loading efficiency when the calcium carbonate has a rich porous structure as a carrier.
The gelatin and the chitosan are macromolecules of natural sources, have good water absorption, biocompatibility and sustainability, and can form a framework structure on the surface of the calcium carbonate microsphere through self-assembly of intermolecular hydrogen bonds and electrostatic interaction.
The preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5-6 to obtain a mixed solution a; mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL gelatin aqueous solution to obtain mixed solution b;
s3, functionalized calcium carbonate microspheres: and adding all the calcium carbonate microspheres obtained in the step S1 into all the mixed liquor a obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, centrifuging at 8000rpm for 5-15min after the completion, adding the precipitate into all the mixed liquor b obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, adding 8-15 parts by weight of a cross-linking agent, continuously stirring for 15-30min, and centrifuging, washing and drying after the completion to obtain the functionalized calcium carbonate microspheres.
Fulvic acid is an excellent soil conditioner, can improve the drought resistance of crops, promote nutrient absorption, stabilize the pH value of soil, reduce fertilizer loss and provide multiple benefits for crop growth; the magnesium ammonium phosphate is rich in N, P, Mg, has good biocompatibility, can be used as slow release fertilizer, and reacts with heavy metal in soil to form highly insoluble metal-containing hydroxyapatite. The functionalized calcium carbonate microspheres prepared by the invention have high loading capacity on fulvic acid and magnesium ammonium phosphate, good slow release performance and excellent biodegradability.
Preferably, the preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5-6 to obtain a mixed solution a; mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL gelatin aqueous solution to obtain mixed solution b;
s3, adding all the calcium carbonate microspheres obtained in the step S1 into 40-60 parts by weight of mixed solution c, and treating for 5-15min in ultrasonic waves with the power of 150-250W and the frequency of 30-50kHz to obtain a turbid solution; the mixed solution c consists of 2-5mg/mL fulvic acid aqueous solution and 0.1-0.5mg/mL magnesium ammonium phosphate aqueous solution according to the mass ratio of 1: 1;
s4, functionalized calcium carbonate microspheres: and adding all the turbid liquid obtained in the step S3 into all the mixed liquid a obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, centrifuging at 8000rpm for 5-15min after the stirring is finished, adding the precipitate into all the mixed liquid b obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, adding 8-15 parts by weight of cross-linking agent, continuously stirring for 15-30min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
In the present invention, firstly hydroxyethylcellulose is used as a regulator, passing Ca2+With CO3 2-The calcium carbonate microspheres with uniform size are prepared through reaction, a large number of carboxyl groups are modified on the surfaces of the microspheres through hydroxyethyl cellulose, then, micromolecular fulvic acid and ammonium magnesium phosphate are absorbed by utilizing capillary force provided by micropores on the surfaces of the calcium carbonate, then, the gelatin and the chitosan complete a self-assembly process on the surfaces of the calcium carbonate microspheres through electrostatic adsorption and intermolecular interaction, and a cross-linking agent and the polyelectrolyte are used for cross-linking to remove excessive polyelectrolyte, so that a microsphere structure with the characteristics of biodegradability and sustained release is finally formed.
The cross-linking agent is glutaraldehyde and/or malonic acid.
Preferably, the cross-linking agent is a mixture consisting of 1 wt% of glutaraldehyde aqueous solution and 1 wt% of malonic acid aqueous solution according to the mass ratio of 1 (1-2).
The invention uses the combination of glutaraldehyde and malonic acid as a cross-linking agent, because aldehyde groups in the glutaraldehyde and carboxyl groups in the malonic acid can react with amino groups on gelatin and chitosan, excessive polyelectrolyte in the solution is removed, and meanwhile, the malonic acid can also make the structure on the surface of the microsphere rougher and generate more irregular hollow structures inside, thereby increasing the specific surface area of the microsphere and improving the adsorption capacity of the zymocyte liquid.
The invention utilizes the functionalized calcium carbonate microspheres as a carrier for fixing microorganisms, the functionalized calcium carbonate microspheres are inorganic-organic functional synergistic substances which are rough in surface, full of microporous structures in the interior, excellent in adsorption performance and biodegradable, and are friendly carriers for carrying microorganisms, on one hand, the gelatin and chitosan on the surface of the functionalized calcium carbonate microspheres have a large number of positive charges, and can adsorb negatively charged microorganisms through electrostatic action, on the other hand, the functionalized calcium carbonate microspheres have a large number of pore structures in the interior, so that a part of the microorganisms can enter the interior of the microspheres through capillary effect, and the microorganisms in the interior can be slowly released along with the degradation of the microspheres, thereby achieving the purpose of continuous action of the microbial agent.
The invention has the beneficial effects that: the invention provides an efficient microbial agent for solving continuous cropping obstacles, which solves the problems that the microbial agent in the prior art is low in microbial load and cannot continuously exert the effect of a microbial agent due to the fact that the microbial agent is adsorbed by carriers such as earthworm dung, zeolite and the like. The efficient microbial agent for solving the continuous cropping obstacles, prepared by the invention, not only can promote the growth and development of continuous cropping obstacles, but also can improve the soil generated by the continuous cropping obstacles, improve the stress resistance of crops and have wide application prospect.
Detailed Description
The above summary of the present invention is described in further detail below with reference to specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples.
Introduction of some raw materials in this application:
glucose, purchased from huaxin xu chemical technology ltd, su, cat #: HXX-001.
Yeast powder, purchased from yozhou di sha pharmaceutical science ltd, cat #: 2000PPM, active substance content: 70 percent.
Beef extract powder purchased from Beijing Hongrunbao scientific Co., Ltd, cat #: HRBS-Y014A, Specification: and (4) BR.
Bran, available from Shanghai gallop Asahi agricultural products trade company Limited.
Molasses, purchased from Jinnanlongjie chemical company Limited, with effective substance content: 85 percent.
The corn steep liquor is commercially available corn steep liquor dry powder purchased from Jinan Qingyuyuan New Material Co.
Thiobacillus thiooxidans, latin name: acidithiobacillus thiooxidans, strain number: ATCC19377, available from International Logistics center for Biowind.
Calcium carbonate, CAS No.: 471-34-1, available from siemens mezzen bioengineering ltd, active substance content: 99 percent.
Fulvic acid, CAS number: 479-66-3 available from Shanghai Ji to Biochemical technology, Inc.
Magnesium ammonium phosphate, CAS No.: 13478-16-5, available from Shanghai Aladdin Biotechnology Ltd.
Chitosan, CAS No.: 9012-76-4, molecular weight: 10 ten thousand, cargo number: m89296-13107, available from Maya reagents, Inc.
Gelatin, CAS No.: 900-70-8, molecular weight: 5 million, purchased from south Henan brocade Biotech Co.
Example 1
An efficient microbial agent for solving continuous cropping obstacles comprises zymocyte liquid and calcium carbonate.
The preparation method of the efficient microbial agent for solving the continuous cropping obstacle comprises the following steps: mixing the zymophyte liquid and calcium carbonate according to the mass ratio of 1:2, stirring at 800rpm for 30min, and drying at 42 ℃ for 5h to obtain the efficient microbial agent for solving the continuous cropping obstacle.
The preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain with inoculating loop in liquid seed culture medium, culturing at 26 deg.C and 160rpm at constant temperature until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 24 parts of glucose, 18 parts of ammonium sulfate, 12 parts of beef extract powder, 8 parts of yeast powder, 3 parts of potassium chloride, 20 parts of ferrous sulfate and 85 parts of water by weight, wherein the pH value is 3.2, and the beef extract powder is sterilized at 121 ℃ for 20 min;
the strain is thiobacillus thiooxidans;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 10% (V/V), and culturing at constant temperature of 26 ℃ and 160rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 50 parts of molasses, 16 parts of bran, 32 parts of ammonium chloride, 12 parts of corn steep liquor, 23 parts of beef extract powder, 18 parts of yeast powder, 8 parts of sodium chloride, 9 parts of magnesium sulfate, 12 parts of dipotassium hydrogen phosphate, 50 parts of ferrous sulfate and 270 parts of water, wherein the pH value is 3.2, and the beef is sterilized at 121 ℃ for 20 min.
Example 2
An efficient microbial agent for solving continuous cropping obstacles, which consists of zymocyte liquid and calcium carbonate microspheres.
The preparation method of the efficient microbial agent for solving the continuous cropping obstacle comprises the following steps: mixing the zymophyte liquid and the calcium carbonate microspheres according to the mass ratio of 1:2, stirring at 800rpm for 30min, and drying at 42 ℃ for 5h to obtain the efficient microbial agent for solving the continuous cropping obstacle.
The preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain with inoculating loop in liquid seed culture medium, culturing at 26 deg.C and 160rpm at constant temperature until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 24 times the weight of the productGlucose, 18 parts by weight of ammonium sulfate, 12 parts by weight of beef extract powder, 8 parts by weight of yeast powder, 3 parts by weight of potassium chloride, 20 parts by weight of ferrous sulfate and 85 parts by weight of water, wherein the pH value is 3.2, and the beef extract is sterilized at 121 ℃ for 20 min;
the strain is thiobacillus thiooxidans;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 10% (V/V), and culturing at constant temperature of 26 ℃ and 160rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 50 parts of molasses, 16 parts of bran, 32 parts of ammonium chloride, 12 parts of corn steep liquor, 23 parts of beef extract powder, 18 parts of yeast powder, 8 parts of sodium chloride, 9 parts of magnesium sulfate, 12 parts of dipotassium hydrogen phosphate, 50 parts of ferrous sulfate and 270 parts of water, wherein the pH value is 3.2, and the beef is sterilized at 121 ℃ for 20 min.
The preparation method of the calcium carbonate microspheres comprises the following steps: adding 2 parts by weight of hydroxyethyl cellulose into 100 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 20min at room temperature, then continuously adding 100 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 5min, and after the stirring is finished, centrifuging, washing and drying to obtain the calcium carbonate microspheres.
Example 3
An efficient microbial agent for solving continuous cropping obstacles, which consists of zymocyte liquid and functionalized calcium carbonate microspheres.
The preparation method of the efficient microbial agent for solving the continuous cropping obstacle comprises the following steps: mixing zymophyte liquid and functionalized calcium carbonate microspheres according to the mass ratio of 1:2, stirring at 800rpm for 30min, and drying at 42 ℃ for 5h to obtain the efficient microbial agent for solving continuous cropping obstacles.
The preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain with inoculating loop in liquid seed culture medium, culturing at 26 deg.C and 160rpm at constant temperature until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 24 parts by weight of glucose, 18 parts by weight of ammonium sulfate, 12 parts by weight of beef extract powder and 8 parts by weight of beef extract powderYeast powder, 3 parts by weight of potassium chloride, 20 parts by weight of ferrous sulfate and 85 parts by weight of water, wherein the pH value is 3.2, and the mixture is sterilized for 20min at 121 ℃;
the strain is thiobacillus thiooxidans;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 10% (V/V), and culturing at constant temperature of 26 ℃ and 160rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 50 parts of molasses, 16 parts of bran, 32 parts of ammonium chloride, 12 parts of corn steep liquor, 23 parts of beef extract powder, 18 parts of yeast powder, 8 parts of sodium chloride, 9 parts of magnesium sulfate, 12 parts of dipotassium hydrogen phosphate, 50 parts of ferrous sulfate and 270 parts of water, wherein the pH value is 3.2, and the beef is sterilized at 121 ℃ for 20 min.
The preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 2 parts by weight of hydroxyethyl cellulose into 100 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 20min at room temperature, then continuously adding 100 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 5min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5.5 to obtain a mixed solution a; mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL gelatin aqueous solution to obtain a mixed solution b;
s3, functionalized calcium carbonate microspheres: and adding all the calcium carbonate microspheres obtained in the step S1 into all the mixed liquor a obtained in the step S2, stirring for 15min at the temperature of 35 ℃ at 500rpm, centrifuging for 10min at 8000rpm, adding the precipitate into all the mixed liquor b obtained in the step S2, stirring for 15min at 35 ℃ at 500rpm, adding 10 parts by weight of 1 wt% glutaraldehyde aqueous solution, continuing stirring for 20min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
Example 4
An efficient microbial agent for solving continuous cropping obstacles, which consists of zymocyte liquid and functionalized calcium carbonate microspheres.
The preparation method of the efficient microbial agent for solving the continuous cropping obstacle comprises the following steps: mixing zymophyte liquid and functionalized calcium carbonate microspheres according to the mass ratio of 1:2, stirring at 800rpm for 30min, and drying at 42 ℃ for 5h to obtain the efficient microbial agent for solving continuous cropping obstacles.
The preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain with inoculating loop in liquid seed culture medium, culturing at 26 deg.C and 160rpm at constant temperature until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 24 parts of glucose, 18 parts of ammonium sulfate, 12 parts of beef extract powder, 8 parts of yeast powder, 3 parts of potassium chloride, 20 parts of ferrous sulfate and 85 parts of water by weight, wherein the pH value is 3.2, and the beef extract powder is sterilized at 121 ℃ for 20 min;
the strain is thiobacillus thiooxidans;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 10% (V/V), and culturing at constant temperature of 26 ℃ and 160rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 50 parts of molasses, 16 parts of bran, 32 parts of ammonium chloride, 12 parts of corn steep liquor, 23 parts of beef extract powder, 18 parts of yeast powder, 8 parts of sodium chloride, 9 parts of magnesium sulfate, 12 parts of dipotassium hydrogen phosphate, 50 parts of ferrous sulfate and 270 parts of water, wherein the pH value is 3.2, and the beef is sterilized at 121 ℃ for 20 min.
The preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 2 parts by weight of hydroxyethyl cellulose into 100 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 20min at room temperature, then continuously adding 100 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 5min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5.5 to obtain a mixed solution a; mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL gelatin aqueous solution to obtain a mixed solution b;
s3, adding all the calcium carbonate microspheres obtained in the step S1 into 50 parts by weight of the mixed solution c, and treating the mixture for 10min in ultrasonic waves with the power of 200W and the frequency of 40kHz to obtain a turbid solution; the mixed solution c consists of 4mg/mL fulvic acid aqueous solution and 0.5mg/mL magnesium ammonium phosphate aqueous solution according to the mass ratio of 1: 1;
s4, functionalized calcium carbonate microspheres: and adding the whole turbid liquid obtained in the step S3 into the whole mixed liquid a obtained in the step S2, stirring for 15min at the temperature of 35 ℃ at 500rpm, centrifuging for 10min at 8000rpm, adding the precipitate into the whole mixed liquid b obtained in the step S2, stirring for 15min at 35 ℃ at 500rpm, adding 10 parts by weight of 1 wt% glutaraldehyde aqueous solution, continuing stirring for 20min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
Example 5
Essentially the same as example 4, except that:
the preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 2 parts by weight of hydroxyethyl cellulose into 100 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 20min at room temperature, then continuously adding 100 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 5min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5.5 to obtain a mixed solution a; mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL gelatin aqueous solution to obtain a mixed solution b;
s3, adding all the calcium carbonate microspheres obtained in the step S1 into 50 parts by weight of the mixed solution c, and treating the mixture for 10min in ultrasonic waves with the power of 200W and the frequency of 40kHz to obtain a turbid solution; the mixed solution c consists of 4mg/mL fulvic acid aqueous solution and 0.5mg/mL magnesium ammonium phosphate aqueous solution according to the mass ratio of 1: 1;
s4, functionalized calcium carbonate microspheres: and adding the whole turbid liquid obtained in the step S3 into the whole mixed liquid a obtained in the step S2, stirring for 15min at the temperature of 35 ℃ at 500rpm, centrifuging for 10min at 8000rpm, adding the precipitate into the whole mixed liquid b obtained in the step S2, stirring for 15min at 35 ℃ at 500rpm, adding 10 parts by weight of 1 wt% malonic acid aqueous solution, continuing stirring for 20min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
Example 6
Essentially the same as example 4, except that:
the preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 2 parts by weight of hydroxyethyl cellulose into 100 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 20min at room temperature, then continuously adding 100 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 5min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5.5 to obtain a mixed solution a; mixing 50 parts by weight of 0.5mol/L potassium chloride aqueous solution with 50 parts by weight of 2mg/mL gelatin aqueous solution to obtain a mixed solution b;
s3, adding all the calcium carbonate microspheres obtained in the step S1 into 50 parts by weight of the mixed solution c, and treating the mixture for 10min in ultrasonic waves with the power of 200W and the frequency of 40kHz to obtain a turbid solution; the mixed solution c consists of 4mg/mL fulvic acid aqueous solution and 0.5mg/mL magnesium ammonium phosphate aqueous solution according to the mass ratio of 1: 1;
s4, functionalized calcium carbonate microspheres: and adding the whole turbid solution obtained in the step S3 into the whole mixed solution a obtained in the step S2, stirring for 15min at the temperature of 35 ℃ at 500rpm, centrifuging for 10min at 8000rpm, adding the precipitate into the whole mixed solution b obtained in the step S2, stirring for 15min at 35 ℃ at 500rpm, adding 5 parts by weight of 1 wt% glutaraldehyde aqueous solution and 5 parts by weight of 1 wt% malonic acid aqueous solution, continuing stirring for 20min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
Test example 1
Testing the growth condition of the muskmelon at the seedling stage:
(1) loading soil and applying base fertilizer: and (3) naturally airing cold-shed plough layer soil for continuously planting 6-year melons, and screening by using a 0.2mm mesh screen to obtain a test soil sample. Test soil samples with the same weight are taken as test groups 1-6 and a control group respectively, and each group is divided into three parts in parallel. Wherein the high-efficiency microbial inoculum prepared in the examples 1-6 for solving the continuous cropping obstacle is added into the test groups 1-6 and is uniformly mixed, and the addition amount of the microbial inoculum is 5% (W/W); then respectively putting the materials into plastic flowerpots with the same specification, wherein the specifications are marked as L1, L2, L3, L4, L5 and L6, and the reference group is not added with a microbial agent and is marked as CK;
(2) sowing and cultivating: soaking melon seeds (variety Boyang No. 9) in 45 deg.C warm water for 2h, sowing 10 seeds in each pot, setting the greenhouse culture conditions at 26 deg.C, humidity 55%, natural illumination intensity 60%, illumination time 12h per day, counting emergence rate when 2 cotyledons grow out, thinning, and reserving 5 seedlings with consistent growth vigor in each pot;
(3) the growth of the melon at the seedling stage was measured after 20 days, and the results are shown in table 1.
Table 1: emergence rate and growth condition of muskmelon at seedling stage
| Rate of emergence (%) | Plant height (cm) | Stem diameter (mm) | Fresh weight of plant (g) | |
| CK | 46.7 | 14.80 | 2.34 | 29.74 |
| L1 | 66.7 | 16.91 | 2.52 | 33.58 |
| L2 | 76.7 | 20.64 | 2.76 | 37.26 |
| L3 | 83.3 | 25.13 | 3.15 | 40.69 |
| L4 | 90.0 | 30.57 | 3.57 | 43.81 |
| L5 | 93.3 | 30.72 | 3.59 | 43.85 |
| L6 | 96.7 | 32.45 | 3.76 | 46.13 |
The results show that the emergence rate and the growth condition of the muskmelon in the seedling stage of the muskmelon to which the microbial inoculum is applied are obviously superior to those of a blank group, so that the microbial inoculum prepared by the invention can promote the growth indexes of germination, plant height, stem thickness, fresh weight of plants and the like of crops to be effectively increased; further comparing L1-L4, it can be seen that compared with the calcium carbonate of L1, L2-L4 uses calcium carbonate microspheres as carriers of the zymophyte liquid, and has more obvious promotion effect on the growth of melon in seedling stage, probably because the specific surface area of the calcium carbonate microspheres is larger, the adsorption capacity of the zymophyte liquid is increased; compared with the L3 method of directly self-assembling gelatin and chitosan on the surface of calcium carbonate, the L4 carrier functionalized calcium carbonate microsphere of the microbial agent firstly adsorbs micromolecular organic matters into the calcium carbonate microsphere through capillary force, then self-assembles the gelatin and the chitosan on the surface of the calcium carbonate microsphere through electrostatic adsorption and intermolecular interaction, a part of soil conditioner such as micromolecular organic matters is added into the functionalized calcium carbonate microsphere, and when the zymocyte liquid plays a role, the micromolecular substances in the carrier are gradually released into soil along with the degradation of the carrier, so that the soil structure is improved, and growth substances are provided for crops, therefore, the growth indexes such as plant height, stem thickness, fresh plant weight and the like of the melon seedlings are effectively improved.
Test example 2
And (3) measuring the physical and chemical properties of the soil:
(1) loading soil and applying base fertilizer: and (3) naturally airing cold-shed plough layer soil for continuously planting 6-year melons, and screening by using a 0.2mm mesh screen to obtain a test soil sample. Test soil samples with the same weight are taken as test groups 1-6 and a control group respectively, and each group is divided into three parts in parallel. Wherein the high-efficiency microbial inoculum prepared in the examples 1-6 for solving the continuous cropping obstacle is added into the test groups 1-6 and is uniformly mixed, and the addition amount of the microbial inoculum is 5% (W/W); then respectively putting the materials into plastic flowerpots with the same specification, wherein the specifications are marked as L1, L2, L3, L4, L5 and L6, and the reference group is not added with a microbial agent and is marked as CK;
(2) sowing and cultivating: soaking melon seeds (variety Boyang No. 9) in 45 deg.C warm water for 2h, sowing 10 seeds in each pot, setting the greenhouse culture conditions at 26 deg.C, humidity 55%, natural illumination intensity 60%, illumination time 12h per day, thinning when 2 cotyledons grow out, and reserving 5 seedlings with consistent growth vigor in each pot;
(3) and (3) measuring the property of the potted soil after 20 days, collecting the soil in each group of melon planting pots, naturally airing the soil, and sieving the soil through a sieve with the aperture of 0.25mm to obtain an air-dried soil sample to be measured.
1. The content of organic matters in soil is as follows: the determination is carried out by referring to DB 12/T961-.
The specific method comprises the following steps: accurately weighing 0.2500g of air-dried soil sample to be detected in each group of embodiments, placing the air-dried soil sample into a glass digestion tube, adding 10.00mL of 0.4mol/L potassium dichromate-sulfuric acid solution, covering a tube plug, placing the digestion tube into a porous furnace, keeping the temperature in the tube at 175 ℃, heating until the solution in the tube boils and keeps for 5min, taking out the digestion tube, cooling for 30min, transferring the digestion solution and soil residues into a 250mL triangular flask with water without damage, washing the tube and the tube plug with water, merging washing liquid into the triangular flask, and controlling the total volume of the solution in the triangular flask to be 50-60 mL. Adding 3 drops of phenanthroline indicator, titrating with 0.1mol/L ferrous sulfate standard solution, and finally changing the solution from orange to blue-green to brownish-red to reach the end point.
2. Effective phosphorus content: the available phosphorus of the soil in each group of examples is measured according to HJ 704-2014 sodium bicarbonate leaching-molybdenum antimony anti-spectrophotometry for determining available phosphorus in the soil, and the test results are shown in Table 2.
Table 2: soil physical and chemical property test result
The thiobacillus thiooxidans is used as a strain of a zymocyte liquid, and the strain can release organic acid (such as tartaric acid, acetic acid and oxalic acid) to the outside of cells by secreting phytase, nuclease, dehydrogenase, phosphatase and the like so as to acidify the insoluble phosphorus in the continuous cropping soil and convert the insoluble phosphorus into effective phosphorus which is beneficial to plant absorption; therefore, compared with a blank group, the microbial agent prepared by the invention effectively improves the content of organic matters and available phosphorus in the continuous cropping soil. Compared with L3 and L4-6, the content of available phosphorus in L3 is obviously lower than that of L4-6, which is probably because the carrier of the microbial agent in L4-6 absorbs a part of small molecular organic matter, namely magnesium ammonium phosphate, and the contained phosphorus is gradually released into the soil along with the degradation of the carrier, so that the content of available phosphorus in the soil is increased.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Claims (9)
1. The efficient microbial agent for solving the continuous cropping obstacle is characterized by consisting of a zymocyte liquid and a carrier.
2. The efficient microbial inoculant for solving the continuous cropping obstacles as claimed in claim 1, wherein the preparation method of the zymocyte liquid comprises the following steps:
(1) inoculating the strain picked by the inoculating loop into a liquid seed culture medium, and culturing at constant temperature of 26 ℃ and 150-180rpm until the thallus concentration reaches 108cfu/mL to obtain a seed solution; the liquid seed culture medium is as follows: 20-26 parts of glucose, 15-20 parts of ammonium sulfate, 10-14 parts of beef extract powder, 6-9 parts of yeast powder, 2-4 parts of potassium chloride, 18-25 parts of ferrous sulfate and 80-90 parts of water, wherein the pH value is 3-3.5, and the beef extract is sterilized at the temperature of 120-;
(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium according to the inoculation amount of 8-12% (V/V), and culturing at constant temperature of 26 ℃ and under the condition of 150-180rpm until the thallus concentration reaches 1010cfu/mL to obtain a zymogen liquid; the fermentation medium is as follows: 45-55 parts of molasses, 14-18 parts of bran, 30-35 parts of ammonium chloride, 10-14 parts of corn steep liquor, 21-24 parts of beef extract powder, 15-20 parts of yeast powder, 5-10 parts of sodium chloride, 7-10 parts of magnesium sulfate, 10-15 parts of dipotassium hydrogen phosphate, 48-57 parts of ferrous sulfate and 260 parts of water, wherein the pH value is 3-3.5, and the materials are sterilized at 125 ℃ for 15-25 min.
3. The efficient microbial inoculant for solving the continuous cropping obstacle as claimed in claim 1, wherein the carrier is any one of diatomite, zeolite, medical stone, calcium carbonate microspheres and functionalized calcium carbonate microspheres.
4. The efficient microbial inoculant for solving the continuous cropping obstacle as claimed in claim 3, wherein the carrier is calcium carbonate microspheres or functionalized calcium carbonate microspheres.
5. The efficient microbial inoculant for solving the continuous cropping obstacle as claimed in claim 4, wherein the calcium carbonate microspheres are prepared by the following steps: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain the calcium carbonate microspheres.
6. The efficient microbial agent for solving the continuous cropping obstacle as claimed in claim 4, wherein the preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5-6 to obtain a mixed solution a; mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL gelatin aqueous solution to obtain mixed solution b;
s3, functionalized calcium carbonate microspheres: and adding all the calcium carbonate microspheres obtained in the step S1 into all the mixed liquor a obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, centrifuging at 8000rpm for 5-15min after the completion, adding the precipitate into all the mixed liquor b obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, adding 8-15 parts by weight of a cross-linking agent, continuously stirring for 15-30min, and centrifuging, washing and drying after the completion to obtain the functionalized calcium carbonate microspheres.
7. The efficient microbial agent for solving the continuous cropping obstacle as claimed in claim 4, wherein the preparation method of the functionalized calcium carbonate microspheres comprises the following steps:
s1, preparation of calcium carbonate microspheres: adding 1-3 parts by weight of hydroxyethyl cellulose into 90-110 parts by weight of 0.1mol/L calcium chloride aqueous solution, stirring at the rotating speed of 1200rpm for 15-25min at room temperature, then continuously adding 90-110 parts by weight of 0.1mol/L sodium carbonate aqueous solution, stirring at the rotating speed of 1000rpm at room temperature for 3-8min, and after the stirring, centrifuging, washing and drying to obtain calcium carbonate microspheres;
s2, mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL chitosan aqueous solution, and adjusting the pH value to 5-6 to obtain a mixed solution a; mixing 40-60 parts by weight of 0.5mol/L potassium chloride aqueous solution and 40-60 parts by weight of 2mg/mL gelatin aqueous solution to obtain mixed solution b;
s3, adding all the calcium carbonate microspheres obtained in the step S1 into 40-60 parts by weight of mixed solution c, and treating for 5-15min in ultrasonic waves with the power of 150-250W and the frequency of 30-50kHz to obtain a turbid solution; the mixed solution c consists of 2-5mg/mL fulvic acid aqueous solution and 0.1-0.5mg/mL magnesium ammonium phosphate aqueous solution according to the mass ratio of 1: 1;
s4, functionalized calcium carbonate microspheres: and adding all the turbid liquid obtained in the step S3 into all the mixed liquid a obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, centrifuging at 8000rpm for 5-15min after the stirring is finished, adding the precipitate into all the mixed liquid b obtained in the step S2, stirring at 500rpm for 10-20min at the temperature of 35 ℃, adding 8-15 parts by weight of cross-linking agent, continuously stirring for 15-30min, and centrifuging, washing and drying after the stirring is finished to obtain the functionalized calcium carbonate microspheres.
8. The efficient microbial inoculant for solving the continuous cropping obstacle as claimed in claim 6 or 7, wherein the cross-linking agent is glutaraldehyde and/or malonic acid.
9. The method for preparing a highly effective microbial inoculum for solving continuous cropping obstacles as claimed in any one of claims 1-8, comprising the steps of: mixing the zymocyte liquid and the carrier according to the mass ratio of 1 (1-3), stirring at the speed of 600-1000rpm for 20-40min, drying at the temperature of 40-45 ℃ for 4-6h, crushing, and sieving with a 80-mesh sieve to obtain the efficient microbial agent for solving the continuous cropping obstacle.
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